Detoxifying pre-treated lignocellulose-containing materials

ABSTRACT

The invention relates to a process of detoxifying pre-treated lignocellulose-containing material comprising subjecting the pre-treated lignocellulose-containing material to one or more phenolic compound oxidizing enzymes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofinternational application no. PCT/US2008/60766 filed Apr. 18, 2008,which claims priority or the benefit under 35 U.S.C. 119 of U.S.provisional application Nos. 60/988,949, 60/946,272 and 60/913,581 filedNov. 9, 2007, Jun. 26, 2007 and Apr. 24, 2007 the contents of which arefully incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to processes of detoxifying pre-treatedlignocellulose-containing material. The invention also relates toprocesses of producing a fermentation product fromlignocellulose-containing material using a fermenting organism includinga detoxification process of the invention.

BACKGROUND OF THE INVENTION

Due to the limited reserves of fossil fuels and worries about emissionof greenhouse gases there is an increasing focus on using renewableenergy sources. Production of fermentation products fromlignocellulose-containing material is known in the art andconventionally includes pretreatment, hydrolysis, and fermentation ofthe lignocellulose-containing material. Pre-treatment results in therelease of, e.g., phenolics and furans, from thelignocellulose-containing material that may irreversibly bind enzymesadded during hydrolysis and fermentation. These compounds may also betoxic to the fermenting organism's metabolism and inhibit theperformance of the fermenting organism.

Detoxification by steam stripping has been suggested but it is acumbersome and a costly additional process step. It has also beensuggested to wash the pre-treated lignocellulose-containing materialbefore hydrolysis. This requires huge amounts of water, that needs to beremoved again, and is therefore also costly.

Consequently, there is a need for providing processes for detoxifyingpre-treated lignocellulose-containing material suitable for fermentationproduct production processes.

SUMMARY OF THE INVENTION

The present invention relates to processes of detoxifying pre-treatedlignocellulose-containing material. The invention also relates toprocesses of producing a fermentation product fromlignocellulose-containing material using a fermenting organism includinga detoxification process of the invention.

In the first aspect the invention relates to processes for detoxifyingpre-treated lignocellulose-containing material comprising subjecting thepre-treated lignocellulose-containing material to one or more phenoliccompound oxidizing enzymes and/or one or more enzymes exhibitingperoxidase activity.

In the second aspect the invention relates to processes for producing afermentation product from lignocellulose-containing material comprisingthe steps of:

(a) pre-treating lignocellulose-containing material;

(b) hydrolyzing;

(c) detoxifying in accordance with a detoxification process of theinvention; and

(d) fermenting using a fermenting organism.

The invention also relates to processes for producing a fermentationproduct from lignocellulose-containing material comprising the steps of:

(i) pre-treating lignocellulose-containing material;

(ii) detoxifying in accordance with the fermentation process of theinvention;

(iii) hydrolyzing; and

(iv) fermenting using a fermenting organism.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of laccase treatment on cellulose conversion.

DETAILED DESCRIPTION OF THE INVENTION

In the first aspect the invention relates to processes of detoxifyingpre-treated lignocellulose-containing material suitable for producing afermentation product.

Lignocellulose-Containing Material

The term “lignocellulose-containing materials” used herein refers tomaterial that primarily consists of cellulose, hemicellulose, andlignin. Such material is often referred to as “biomass”.

The structure of lignocellulose is not directly accessible to enzymatichydrolysis. Therefore, the lignocellulose has to be pre-treated, e.g.,by acid hydrolysis under adequate conditions of pressure andtemperature, in order to break the lignin seal and disrupt thecrystalline structure of cellulose. This causes solubilization andsaccharification of the hemicellulose fraction. The cellulose fractioncan then be hydrolyzed enzymatically, e.g., by cellulase enzymes (orcellulolytic enzymes), to convert the carbohydrate polymers intofermentable sugars which may be fermented into a desired fermentationproduct, such as ethanol. Optionally the fermentation product isrecovered after fermentation, e.g., by distillation.

Any lignocellulose-containing material is contemplated according to thepresent invention. The lignocellulose-containing material may be anymaterial containing lignocellulose. In a preferred embodiment thelignocellulose-containing material contains at least 30 wt. %,preferably at least 50 wt. %, more preferably at least 70 wt. %, evenmore preferably at least 90 wt. % lignocellulose. It is to be understoodthat the lignocellulose-containing material may also comprise otherconstituents such as cellulosic material, including cellulose andhemicellulose, and may also comprise other constituents such asproteinaceous material, starch, sugars, such as fermentable sugarsand/or un-fermentable sugars.

Lignocellulose-containing material is generally found, for example, inthe stems, leaves, hulls, husks, and cobs of plants or leaves, branches,and wood of trees. Lignocellulose-containing material can also be, butis not limited to, herbaceous material, agricultural residues, forestryresidues, municipal solid wastes, waste paper, and pulp and paper millresidues. It is understood herein that lignocellulose-containingmaterial may be in the form of plant cell wall material containinglignin, cellulose, and hemi-cellulose in a mixed matrix.

In a preferred embodiment the lignocellulose-containing material is cornfiber, rice straw, pine wood, wood chips, poplar, bagasse, paper andpulp processing waste.

Other examples include corn stover, hardwood, such as poplar and birch,softwood, cereal straw, such as wheat straw, switchgrass, municipalsolid waste (MSW), industrial organic waste, office paper, or mixturesthereof.

In a preferred embodiment the lignocellulose-containing material is cornstover. In another preferred embodiment the material is corn fiber.

Process of Detoxifying Pre-Treated Lignocellulose-Containing Material

When lignocellulose-containing material is pre-treated, degradationproducts that may inhibit enzymes and/or may be toxic to fermentingorganisms are produced. These degradation products severely decreaseboth the hydrolysis and fermentation rate.

Methods for pre-treating lignocellulose-containing material are wellknown in the art. Examples of contemplated methods are described belowin the section “Pre-treatment”.

The present inventors have found that phenolic compound oxidizingenzymes can be used to detoxify pre-treated lignocellulose-containingmaterial. The fermentation time can be reduced as a result of improvedperformance of the fermenting organism during fermentation. In otherwords, detoxification in accordance with the invention may result in ashorter “lignocellulose-containing material-to-fermentation product”process time. Furthermore, the need for a washing step afterpre-treatment of the lignocellulose-containing material, to remove toxiccompounds, and/or adaption of the fermentation organism to themedium/broth can be eliminated. Also, the dosing of the fermentationorganism may be reduced.

In a preferred embodiment the pre-treated lignocellulose-containingmaterial may be treated with cellulase (cellulolytic enzymes) and/orhemicellulase (hemicellulolytic enzymes).

Specific examples of detoxifying compounds can be found in the“Detoxifying Compounds”-section below.

In the first aspect the invention relates to processes for detoxifyingpre-treated lignocellulose-containing material comprising subjecting thepre-treated lignocellulose-containing material to one or more phenoliccompound oxidizing enzymes and/or one or more enzymes exhibitingperoxidase activity.

The pre-treated lignocellulose degradation products include lignindegradation products, cellulose degradation products and hemicellulosedegradation products. The pre-treated lignin degradation products may bephenolics in nature.

The hemicellulose degradation products include furans from sugars (suchas hexoses and/or pentoses), including xylose, mannose, galactose,rhamanose, and arabinose. Examples of hemicelluloses include xylan,galactoglucomannan, arabinogalactan, arabinoglucuronoxylan,glucuronoxylan, and derivatives and combinations thereof.

Examples of inhibitory compounds, i.e., pre-treated lignocellulosedegradation products, include 4-OH benzyl alcohol, 4-OH benzaldehyde,4-OH benzoic acid, trimethyl benzaldehyde, 2-furoic acid, coumaric acid,ferulic acid, phenol, guaiacol, veratrole, pyrogallollol, pyrogallolmono methyl ether, vanillyl alcohol, vanillin, isovanillin, vanillicacid, isovanillic acid, homovanillic acid, veratryl alcohol,veratraldehyde, veratric acid, 2-O-methyl gallic acid, syringyl alcohol,syringaldehyde, syringic acid, trimethyl gallic acid, homocatechol,ethyl vanillin, creosol, p-methyl anisol, anisaldehyde, anisic acid,furfural, hydroxymethylfurfural, 5-hydroxymethylfurfural, formic acid,acetic acid, levulinic acid, cinnamic acid, coniferyl aldehyde,isoeugenol, hydroquinone, eugenol or combinations thereof. Otherinhibitory compounds can be found in, e.g., Luo et al., 2002, Biomassand Bioenergy 22: 125-138.

The detoxification process of the invention may preferably be carriedout at a pH that is suitable of the phenolic compound oxidizing enzymesand hydrolyzing enzyme(s) and/or fermenting organism if detoxificationis carried out simultaneously with hydrolysis or simultaneously withhydrolysis and fermentation. In one embodiment the pH is between 2 and7, preferably between 3 and 6, especially between 4 and 5. In apreferred embodiment the temperature during detoxification is atemperature suitable for the phenolic compound oxidizing enzyme(s)and/or enzyme exhibiting peroxidase activity and hydrolyzing enzyme(s)and/or fermenting organism if detoxification is carried out asimultaneous with hydrolysis or simultaneously with hydrolysis andfermentation. In one embodiment the temperature during detoxification isbetween 25° C. and 70° C., preferably between 30° C. and 60° C. In caseswhere detoxification is carried out simultaneously with fermentation thetemperature will depend on the fermenting organism. For ethanolfermentations with yeast the temperature would be between 26-38° C.,such as between 26-34° C. or between 30-36° C., such as around 32° C.

Suitable pHs, temperatures and other process conditions can easily bedetermined by one skilled in the art.

Detoxifying Enzymes

The detoxifying enzyme(s) may be of any origin including of mammal,plant and microbial origin, such as of bacteria and fungal origin.

Phenolic compound oxidizing enzymes may in preferred embodiments belongto any of the following EC classes including: Catechol oxidase (EC1.10.3.1), Laccase (EC 1.10.3.2), o-Aminophenol oxidase (1.10.3.4); andMonophenol monooxygenase (1.14.18.1).

The enzyme exhibiting peroxidase activity may in a preferred embodimentbelong to any of the following EC classes including those selected fromthe group consisting of a peroxidase (EC 1.11.1.7), Haloperoxidase(EC1.11.1.8 and EC 1.11.1.10); Lignin peroxidase (EC 1.11.1.14);manganese peroxidase (EC 1.11.1.13); and Lipoxygenase (EC.1.13.11.12).

Examples of detoxifying enzymes contemplated according to the inventioncan be found in the “Enzymes”-section below.

Production of Fermentation Products from Lignocellulose-ContainingMaterial

In the second aspect the invention relates to processes of producingfermentation products from lignocellulose-containing material.Conversion of lignocellulose-containing material into fermentationproducts, such as ethanol, has the advantages of the ready availabilityof large amounts of feedstock, including wood, agricultural residues,herbaceous crops, municipal solid wastes, etc.

The structure of lignocellulose is not directly accessible to enzymatichydrolysis. Therefore, the lignocellulose-containing material has to bepre-treated, e.g., by acid hydrolysis under adequate conditions ofpressure and temperature, in order to break the lignin seal and disruptthe crystalline structure of cellulose. This causes solubilization ofthe hemicellulose and cellulose fractions. The cellulose andhemicellulose can then be hydrolyzed enzymatically, e.g., by cellulaseenzymes (cellulolytic enzymes), to convert the carbohydrate polymersinto fermentable sugars which may be fermented into a desiredfermentation product, such as ethanol. Optionally the fermentationproduct may be recovered, e.g., by distillation.

More precisely the invention relates in this embodiment to processes forproducing a fermentation product from lignocellulose-containing materialcomprising the steps of:

(a) pre-treating lignocellulose-containing material;

(b) hydrolyzing;

(c) detoxifying; and

(d) fermenting using a fermenting organism;

wherein detoxification is carried out in accordance with adetoxification process of the invention. More details on the steps aredescribed below in the sections “Pre-treatment”, “Hydrolysis” and“Fermentation”.

In another embodiment the invention relates to processes for producing afermentation product from lignocellulose-containing material comprisingthe steps of:

(i) pre-treating lignocellulose-containing material;

(ii) detoxifying;

(iii) hydrolyzing; and

(iv) fermenting using a fermenting organism;

wherein detoxification is carried out in accordance with adetoxification process of the invention.

One or more detoxifying enzymes may be added after pre-treatment step(i), but before hydrolysis step (iii). Detoxifying the pre-treatedmaterial in step (ii) may be carried out before hydrolysis, butdetoxification and hydrolysis may also be carried out simultaneously.The detoxification step (ii) may be carried out separately fromhydrolysis. Further hydrolysis step (iii) and fermentation step (iv) maybe carried out simultaneously or sequentially. In one embodiment thesolids (comprising mainly lignin and unconverted polysaccharides) may,after pre-treating the lignocellulose-containing material in step (i),be removed/separated from the liquor before detoxification. The removedsolids and the detoxified liquor may be combined before hydrolysis instep (iii) or simultaneous hydrolysis and fermentation.

The solids may be removed/separated in any suitable way know in the art.In suitable embodiments the solids are removed by filtration, or byusing a filter press and/or centrifuge, or the like. The reducedinhibitory effect of the hydrolyzing enzymes is tested in Example 4.

Examples of pre-treatment methods, hydrolysis and fermentationconditions for both of above embodiment is described below.

Pre-Treatment

The lignocellulose-containing material may be pre-treated in anysuitable way. Pre-treatment may be carried out before and/or duringhydrolysis and/or fermentation. In a preferred embodiment thepre-treated material is hydrolyzed, preferably enzymatically, beforeand/or during fermentation and/or before and/or during detoxification.The goal of pre-treatment is to separate and/or release cellulose;hemicellulose and/or lignin and this way improve the rate of hydrolysis.Pre-treatment methods such as wet-oxidation and alkaline pre-treatmenttargets lignin, while dilute acid and auto-hydrolysis targetshemicellulose. Steam explosion is an example of a pre-treatment thattargets cellulose.

According to the invention pre-treatment in step (a) or (i) may be aconventional pre-treatment step using techniques well known in the art.Examples of suitable pre-treatments are disclosed below. In a preferredembodiment pre-treatment takes place in aqueous slurry.

The lignocellulose-containing material may during pre-treatment bepresent in an amount between 10-80 wt. %, preferably between 20-70 wt.%, especially between 30-60 wt. %, such as around 50 wt. %.

Chemical, Mechanical and/or Biological Pre-Treatment

The lignocellulose-containing material may according to the invention bechemically, mechanically and/or biologically pre-treated beforehydrolysis and/or fermentation. Mechanical treatment (often referred toas physical treatment) may be used alone or in combination withsubsequent or simultaneous hydrolysis, especially enzymatic hydrolysis.

Preferably, chemical, mechanical and/or biological pre-treatment iscarried out prior to the hydrolysis and/or fermentation. Alternatively,the chemical, mechanical and/or biological pre-treatment may be carriedout simultaneously with hydrolysis, such as simultaneously with additionof one or more cellulase enzymes (cellulolytic enzymes), or other enzymeactivities mentioned below, to release, e.g., fermentable sugars, suchas glucose and/or maltose.

In an embodiment of the invention the pre-treatedlignocellulose-containing material may be washed before and/or afterdetoxification. However, washing is not mandatory and is in a preferredembodiment eliminated.

According to one embodiment of the invention one or more detoxifyingenzymes may be added to the pre-treated lignocellulose-containingmaterial in step (c) or (ii). Detoxification step (c) or (ii) andhydrolysis step (b) or (iii) may be carried out either simultaneously orsequentially. The reduced toxic effect on the fermenting organism isshown in Examples 2 and 3.

The steps may in one embodiment be done in one treating solution (ie.,one bath). In one embodiment the hydrolyzing enzyme(s) and thedetoxifying enzyme(s) are added simultaneously to the treating solution.In another embodiment the hydrolyzing enzyme(s) are added before thedetoxifying enzyme(s). It may be advantageous to complete above 50% ofhydrolysis, preferably above 70% of hydrolysis, especially above 90% ofhydrolysis before adding the detoxifying enzyme(s) to the treatingsolution. If the pre-treated lignocellulose-containing material ishydrolyzed enzymatically, it is advantageous to do detoxification beforeand/or simultaneous with hydrolysis. However, if hydrolysis is carriedout using one or more acids, i.e., acid hydrolysis, detoxification ispreferably carried out after and/or simultaneously with acid hydrolysis.

In another embodiment detoxification step (c) or (ii) may be carried outseparately from hydrolysis step (b) or (iii) and fermentation step (d)or (iv), respectively, which in one embodiment may be carried outsimultaneously. In a further embodiment all of steps (b), (c) and (d) or(i), (ii), (iii) and (iv), respectively, are carried out simultaneouslyor sequentially. When detoxification is done as a separate step, ittypically is carried out for between 1-24 hours.

In a preferred embodiment the pre-treated lignocellulose-containingmaterial is unwashed.

In an embodiment the phenolic compound oxidizing enzyme(s) is(are) dosedin the range from above 0, such as 0.01 to 1 mg/g DS or in the rangefrom above 0 to 100 LACU/g DS. In an embodiment the enzyme(s) exhibitingperoxidase activity is(are) dosed in the range from above 0, such as0.01 to 10 mg/g DS or above 0, such as 0.01 to 100 PODU/g DS.

Chemical Pre-Treatment

The term “chemical treatment” refers to any chemical pre-treatment whichpromotes the separation and/or release of cellulose, hemicelluloseand/or lignin. Examples of suitable chemical pre-treatments includetreatment with; for example, dilute acid, lime, alkaline, organicsolvent, ammonia, sulfur dioxide, carbon dioxide. Further, wet oxidationand pH-controlled hydrothermolysis are also considered chemicalpre-treatment.

In a preferred embodiment the chemical pre-treatment is acid treatment,more preferably, a continuous dilute and/or mild acid treatment, suchas, treatment with sulfuric acid, or another organic acid, such asacetic acid, citric acid, tartaric acid, succinic acid, hydrogenchloride or mixtures thereof. Other acids may also be used. Mild acidtreatment means that the treatment pH lies in the range from 1-5,preferably 1-3. In a specific embodiment the acid concentration is inthe range from 0.1 to 2.0 wt. % acid, preferably sulphuric acid. Theacid may be contacted with the lignocellulose-containing material andthe mixture may be held at a temperature in the range of 160-220° C.,such as 165-195° C., for periods ranging from minutes to seconds, e.g.,1-60 minutes, such as 2-30 minutes or 3-12 minutes. Addition of strongacids, such as sulphuric acid, may be applied to remove hemicellulose.This enhances the digestibility of cellulose.

Other techniques are also contemplated. Cellulose solvent treatment hasbeen shown to convert about 90% of cellulose to glucose. It has alsobeen shown that enzymatic hydrolysis could be greatly enhanced when thelignocellulose structure is disrupted. Alkaline H₂O₂, ozone, organosolv(uses Lewis acids, FeCl₃, (Al)₂SO₄ in aqueous alcohols), glycerol,dioxane, phenol, or ethylene glycol are among solvents known to disruptcellulose structure and promote hydrolysis (Mosier et al., 2005,Bioresource Technology 96: 673-686).

Alkaline chemical pre-treatment with base, e.g., NaOH, Na₂CO₃ and/orammonia or the like, is also contemplated according to the invention.Pre-treatment methods using ammonia are described in, e.g., WO2006/110891, WO 2006/110899, WO 2006/110900, and WO 2006/110901 (whichare hereby incorporated by reference).

Wet oxidation techniques involve use of oxidizing agents, such as:sulphite based oxidizing agents or the like. Examples of solventpre-treatments include treatment with DMSO (Dimethyl Sulfoxide) or thelike. Chemical pre-treatment is generally carried out for 1 to 60minutes, such as from 5 to 30 minutes, but may be carried out forshorter or longer periods of time dependent on the material to bepre-treated.

Other examples of suitable pre-treatment methods are described by Schellet al., 2003, Appl. Biochem and Biotechn. Vol. 105-108: 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Publication No. 2002/0164730, which references are hereby allincorporated by reference.

Mechanical Pre-Treatment

The term “mechanical pre-treatment” refers to any mechanical (orphysical) treatment which promotes the separation and/or release ofcellulose, hemicellulose and/or lignin from lignocellulose-containingmaterial. For example, mechanical pre-treatment includes various typesof milling, irradiation, steaming/steam explosion, and hydrothermolysis.

Mechanical pre-treatment includes comminution (mechanical reduction ofthe size). Comminution includes dry milling, wet milling and vibratoryball milling. Mechanical pre-treatment may involve high pressure and/orhigh temperature (steam explosion). In an embodiment of the inventionhigh pressure means pressure in the range from 300 to 600 psi,preferably 400 to 500 psi, such as around 450 psi. In an embodiment ofthe invention high temperature means temperatures in the range fromabout 100 to 300° C., preferably from about 140 to 235° C. In apreferred embodiment mechanical pre-treatment is a batch-process, steamgun hydrolyzer system which uses high pressure and high temperature asdefined above. A Sunds Hydrolyzer (available from Sunds Defibrator AB(Sweden) may be used for this.

Combined Chemical and Mechanical Pre-Treatment

In a preferred embodiment both chemical and mechanical pre-treatmentsare carried out. For instance, the pre-treatment step may involve diluteor mild acid treatment and high temperature and/or pressure treatment.The chemical and mechanical pre-treatment may be carried outsequentially or simultaneously, as desired.

Accordingly, in a preferred embodiment, the lignocellulose-containingmaterial is subjected to both chemical and mechanical pre-treatment topromote the separation and/or release of cellulose, hemicellulose and/orlignin.

In a preferred embodiment the pre-treatment is carried out as a diluteand/or mild acid steam explosion step. In another preferred embodimentpre-treatment is carried out as an ammonia fiber explosion step (or AFEXpre-treatment step).

Biological Pre-Treatment

As used in the present invention the term “biological pre-treatment”refers to any biological pre-treatment which promotes the separationand/or release of cellulose, hemicellulose, and/or lignin from thelignocellulose-containing material. Biological pre-treatment techniquescan involve applying lignin-solubilizing microorganisms (see, forexample, Hsu, 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, ed., Taylor & Francis, Washington,D.C., 179-212; Ghosh and Singh, 1993, Physicochemical and biologicaltreatments for enzymatic/microbial conversion of lignocellulosicbiomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, 1994, Pretreatinglignocellulosic biomass: a review, in Enzymatic Conversion of Biomassfor Fuels Production, Himmel, Baker, and Overend, eds., ACS SymposiumSeries 566, American Chemical Society, Washington, D.C., chapter 15;Gong, Cao, Du, and Tsao, 1999, Ethanol production from renewableresources, in Advances in Biochemical Engineering/Biotechnology,Scheper, ed., Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241;Olsson and Hahn-Hagerdal, 1996, Fermentation of lignocellulosichydrolysates for ethanol production, Enz. Microb. Tech. 18: 312-331; andVallander and Eriksson, 1990, Production of ethanol from lignocellulosicmaterials: State of the art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Hydrolysis

Before and/or simultaneously with fermentation the pre-treatedlignocellulose-containing material may be hydrolyzed to break downcellulose and hemicellulose.

The dry solids content during hydrolysis may be in the range from 5-50wt. %, preferably 10-40 wt. %, preferably 20-30 wt. %. Hydrolysis may ina preferred embodiment be carried out as a fed batch process where thepre-treated lignocellulose-containing material (substrate) is fedgradually to an, e.g., enzyme containing hydrolysis solution.

In an embodiment of the invention detoxification takes place before,during and/or after hydrolysis.

In a preferred embodiment hydrolysis is carried out enzymatically.According to the invention the pre-treated lignocellulose-containingmaterial may be hydrolyzed by one or more hydrolases (class EC 3according to Enzyme Nomenclature), preferably one or more carbohydrasesselected from the group consisting of cellulase, hemicellulase, amylase,such as alpha-amylase, protease, carbohydrate-generating enzyme, such asglucoamylase, esterase, such as lipase. Alpha-amylase, glucoamylaseand/or the like may be present during hydrolysis and/or fermentation asthe lignocellulose-containing material may include some starch.

The enzyme(s) used for hydrolysis is(are) capable of directly orindirectly converting carbohydrate polymers into fermentable sugarswhich can be fermented into a desired fermentation product, such asethanol.

In a preferred embodiment the carbohydrase has cellulase enzymeactivity. Suitable carbohydrases are described in the “Enzymes”-sectionbelow.

Hemicellulose polymers can be broken down by hemicellulases and/or acidhydrolysis to release its five and six carbon sugar components. The sixcarbon sugars (hexoses), such as glucose, galactose, arabinose, andmannose, can readily be fermented to, e.g., ethanol, acetone, butanol,glycerol, citric acid, fumaric acid, etc. by suitable fermentingorganisms including yeast. Preferred for ethanol fermentation is yeastof the species Saccharomyces cerevisiae, preferably strains which areresistant towards high levels of ethanol, i.e., up to, e.g., about 10,12 or 15 vol. % ethanol or more, such as 20 vol. % ethanol.

In a preferred embodiment the pre-treated lignocellulose-containingmaterial is hydrolyzed using a hemicellulase, preferably a xylanase,esterase, cellobiase, or combination thereof.

Hydrolysis may also be carried out in the presence of a combination ofhemicellulases and/or cellulases, and optionally one or more of theother enzyme activities mentioned in the “Enzyme” section below.

In a preferred embodiment hydrolysis and fermentation is carried out asa simultaneous hydrolysis and fermentation step (SSF). In general thismeans that combined/simultaneous hydrolysis and fermentation are carriedout at conditions (e.g., temperature and/or pH) suitable, preferablyoptimal, for the fermenting organism(s) in question.

In another preferred embodiment hydrolysis and fermentation are carriedout as hybrid hydrolysis and fermentation (HHF). HHF typically beginswith a separate partial hydrolysis step and ends with a simultaneoushydrolysis and fermentation step. The separate partial hydrolysis stepis an enzymatic cellulose saccharification step typically carried out atconditions (e.g., at higher temperatures) suitable, preferably optimal,for the hydrolyzing enzyme(s) in question. The subsequent simultaneoushydrolysis and fermentation step is typically carried out at conditionssuitable for the fermenting organism(s) (often at lower temperaturesthan the separate hydrolysis step). Finally, hydrolysis and fermentationmay also be carried out a separate hydrolysis and fermentation, wherethe hydrolysis is taken to completion before initiation of fermentation.This often referred to as “SHF”.

Enzymatic treatments may be carried out in a suitable aqueousenvironment under conditions which can readily be determined by oneskilled in the art.

In a preferred embodiment hydrolysis is carried out at suitable,preferably optimal conditions for the enzyme(s) in question.

Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art present invention. Preferably,hydrolysis is carried out at a temperature between 25 and 70° C.,preferably between 40 and 60° C., especially around 50° C. The processis preferably carried out at a pH in the range from 3-8, preferably pH4-6, especially around pH 5.

Preferably, hydrolysis is carried out for between 12 and 96 hours,preferable 16 to 72 hours, more preferably between 24 and 48 hours.

According to the invention hydrolysis in step (b) or (iii) andfermentation in step (d) or (iv) may be carried out simultaneously (SSFprocess) or sequentially (SHF process) or as a hybrid hydrolysis andfermentation (HHF).

Fermentation

According to the invention the pre-treated (and hydrolyzed)lignocellulose-containing material is fermented by at least onefermenting organism capable of fermenting fermentable sugars, such asglucose, xylose, mannose, and galactose directly or indirectly into adesired fermentation product.

The fermentation is preferably ongoing for between 8 to 96 hours,preferably 12 to 72 hours, more preferable from 24 to 48 hours.

In an embodiment the fermentation is carried out at a temperaturebetween 20 to 40° C., preferably 26 to 34° C., in particular around 32°C. In an embodiment the pH is from pH 3 to 6, preferably around pH 4 to5.

Contemplated according to the invention is simultaneous hydrolysis andfermentation (SSF). In an embodiment there is no separate holding stagefor the hydrolysis, meaning that the hydrolyzing enzyme(s) and thefermenting organism are added together. When the fermentation (e.g.,ethanol fermentation using Saccharomyces yeast) is performedsimultaneous with hydrolysis the temperature is preferably between 26°C. and 35° C., more preferably between 30° C. and 34° C., such as around32° C. A temperature program comprising at least two holding stages atdifferent temperatures may be applied according to the invention.

The process of the invention may be performed as a batch, fed-batch oras a continuous process.

Recovery

Subsequent to fermentation the fermentation product may be separatedfrom the fermentation medium/broth. The medium/broth may be distilled toextract the fermentation product or the fermentation product may beextracted from the fermentation medium/broth by micro or membranefiltration techniques. Alternatively the fermentation product may berecovered by stripping. Recovery methods are well known in the art.

Fermentation Products

Processes of the invention may be used for producing any fermentationproduct. Especially contemplated fermentation products include alcohols(e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid,acetic acid, itaconic acid, lactic acid, gluconic acid); ketones (e.g.,acetone); amino acids (e.g., glutamic acid); gases (e.g., H₂ and CO₂);antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins(e.g., riboflavin, B12, beta-carotene); and hormones.

Also contemplated products include consumable alcohol industry products,e.g., beer and wine; dairy industry products, e.g., fermented dairyproducts; leather industry products and tobacco industry products. In apreferred embodiment the fermentation product is an alcohol, especiallyethanol. The fermentation product, such as ethanol, obtained accordingto the invention, may preferably be fuel alcohol/ethanol. However, inthe case of ethanol it may also be used as potable ethanol.

Fermenting Organism

The term “fermenting organism” refers to any organism, includingbacterial and fungal organisms, suitable for producing a desiredfermentation product. Especially suitable fermenting organisms accordingto the invention are able to ferment, i.e., convert, sugars, such asglucose, directly or indirectly into the desired fermentation product.Examples of fermenting organisms include fungal organisms, such asyeast. Preferred yeast includes strains of the genus Saccharomyces, inparticular a strain of Saccharomyces cerevisiae or Saccharomyces uvarum;a strain of Pichia, in particular Pichia stipitis or Pichia pastoris; astrain of the genus Candida, in particular a strain of Candida utilis,Candida arabinofermentans, Candida diddensii, or Candida boidinii. Othercontemplated yeast includes strains of Hansenula, in particularHansenula polymorpha or Hansenula anomala; strains of Kluyveromyces, inparticular Kluyveromyces marxianus or Kluyveromyces fagilis, and strainsof Schizosaccharomyces, in particular Schizosaccharomyces pombe.

Preferred bacterial fermenting organisms include strains of Escherichia,in particular Escherichia coli, strains of Zymomonas, in particularZymomonas mobilis, strains of Zymobacter in particular Zymobactorpalmae, strains of Klebsiella, in particular Klebsiella oxytoca, strainsof Leuconostoc, in particular Leuconostoc mesenteroides, strains ofClostridium, in particular Clostridium butyricum, strains ofEnterobacter in particular Enterobacter aerogenes and strains ofThermoanaerobacter, in particular Thermoanaerobacter BG1L1 (Appl.Micrbiol. Biotech. 77: 61-86) and Thermoanarobacter ethanolicus.

Commercially available yeast includes, e.g., RED STAR™ or ETHANOL RED™yeast (available from Fermentis/Lesaffre, USA), FALI (available fromFleischmann's Yeast, USA), SUPERSTART and THERMOSACC™ fresh yeast(available from Ethanol Technology, WI, USA), BIOFERM AFT and XR(available from NABC—North American Bioproducts Corporation, GA, USA),GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL(available from DSM Specialties).

Enzymes

Even though not specifically mentioned in context of processes of theinvention, it is to be understood that the enzymes (as well as othercompounds) are used in an “effective amount”. For instance, “effectiveamount” means, in context of phenolic compound oxidizing enzyme(s) thatit has an improving effect compared to a corresponding process where nophenolic compound oxidizing enzyme(s) was (were) added.

Phenolic Compound Oxidizing Enzymes

Preferred phenolic compound oxidizing enzymes belong to any of thefollowing EC classes: Catechol oxidase (EC 1.10.3.1), Laccase (EC1.10.3.2), o-Aminophenol oxidase (1.10.3.4); and Monophenolmonooxygenase (1.14.18.1).

Laccase

Laccases (EC 1.10.3.2) are multi-copper-containing enzymes that catalyzethe oxidation of phenolic compounds. Laccases are produced by plants,bacteria and also a wide variety of fungi, including Ascomycetes such asAspergillus, Neurospora, and Podospora; Deuteromycete includingBotrytis, and Basidiomycetes such as Collybia, Fomes, Lentinus,Pleurotus, Trametes, and perfect forms of Rhizoctonia. A number offungal laccases have been isolated. For example, Choi et al. (Mol.Plant-Microbe Interactions 5: 119-128, 1992) describe the molecularcharacterization and cloning of the gene encoding the laccase of thechestnut blight fungus, Cryphonectria parasitica. Kojima et al. (J.Biol. Chem. 265: 15224-15230, 1990; JP 2-238885) provide a descriptionof two allelic forms of the laccase of the white-rot basidiomyceteCoriolus hirsutus. Germann and Lerch (Experientia 41: 801, 1985; PNASUSA 83: 8854-8858, 1986) have reported the cloning and partialsequencing of the Neurospora crassa laccase gene. Saloheimo et al. (J.Gen. Microbiol. 137: 1537-1544, 1985; WO 92/01046) have disclosed astructural analysis of the laccase gene from the fungus Phlebia radiata.

Especially contemplated laccases include those derived from a strain ofPolyporus, preferably Polyporus pinsitus; Melanocarpus, preferablyMelanocarpus albomyces; Myceliophtora, preferably Myceliophtorathermophila; Coprinus, preferably Coprinus cinereus; Rhizoctonia,preferably Rhizoctonia solani or Rhizoctonia praticola; Scytalidium,preferably Scytalidium thermophilum; Pyricularia, preferably Pyriculariaoryzae.

In an embodiment the laccase is derived from the tree Rhus vernicifera(Yoshida, 1983, Chemistry of Lacquer (Urushi) part 1. J. Chem. Soc. 43:472-486).

In another embodiment the laccase is derived from Myceliopthorathermophila, e.g., the one described in WO 95/33836 (Novozymes).

In another embodiment the laccase is derived from Polyporus pinsitus,e.g., the one described in WO 96/00290 (Novozymes).

Jonsson et al., 1998, Appl. Microbiol. Biotechnol. 49: 691-697, alsodiscloses a suitable laccase derived from Polyporus versicolar.

Other laccases include the one derived from Pyricularia oryzae concernedin, e.g., Muralikrishna et al., 1995, Appl. Environ. Microbiol. 61(12):4374-4377, or the laccase derived from Scytalidium thermophilum, whichis disclosed in Abstract of Papers American Chemical Society vol. 209,no. 1-2, 1995.

The laccase may also be one derived from Coprinus cinereus, e.g., theone concerned in Schneider et al., 1999, Enzyme and Microbial Technology25: 502-508.

Other suitable laccases include those derived from Rhizoctonia solaniconcerned in Waleithner et al., Curr. Genet., 1996, 29: 395-403, orderived from Melanocarpus albomyces concerned in Kiiskinen et al., 2004,Microbiology 150: 3065-3074.

Suitable bacterial laccase include those derived from Streptomycescoelicolor, e.g., disclosed by Machczynski et al. in Protein Science,2004, 13: 2388-2397.

Enzymes Exhibiting Peroxidase Activity

According to the invention any enzyme exhibiting peroxidase activity maybe used.

The enzyme exhibiting peroxidase activity may be selected from the groupconsisting of a peroxidase (EC 1.11.1.7), haloperoxidase (EC1.11.1.8 andEC 1.11.1.10), lignin perocidase (EC 1.11.1.14), manganese peroxidase(EC 1.11.1.13); and lipoxygenase (EC.1.13.11.12).

Peroxidase

The enzyme exhibiting peroxidase activity may be any peroxidaseclassified as EC 1.11.1.7.

Peroxidases suitable in processes of the invention may be of plant(e.g., horseradish or soybean peroxidase), or microbial origin, such asof fungal or bacteria origin. Examples include peroxidases derived fromfungi of the subdivision Deuteromycotina, class Hyphomycetes, e.g.,Fusarium, Humicola, Tricoderma, Myrothecium, Verticillum, Arthromyces,Caldariomyces, Ulocladium, Embellisia, Cladosporium or Dreschlera, inparticular Fusarium oxysporum (DSM 2672), Humicola insolens, Trichodermareesii, Myrothecium verrucana (IFO 6113), Verticillum alboatrum,Verticillum dahlia, Arthromyces ramosus (FERM P-7754), Caldariomycesfumago, Ulocladium chartarum, Embellisia alli or Dreschlera halodes.

Other suitable peroxidases are derived from fungi including strains ofthe subdivision Basidiomycotina, class Basidiomycetes, e.g., Coprinus,Phanerochaete, Coriolus or Trametes, in particular Coprinus cinereus f.microsporus (IFO 8371), Coprinus macrorhizus, Phanerochaetechrysosporium (e.g., NA-12) or Trametes (previously called Polyporus),e.g., T. versicolor (e.g., PR4 28-A).

Other peroxidases may be derived from fungi including strains belongingto the subdivision Zygomycotina, class Mycoraceae, e.g., Rhizopus orMucor, in particular Mucor hiemalis.

Bacterial peroxidases may be derived from strains of the orderActinomycetales, e.g., Streptomyces spheroides (ATTC 23965),Streptomyces thermoviolaceus (IFO 12382) or Streptoverticillumverticillium ssp. Verticillium; Bacillus pumilus (ATCC 12905), Bacillusstearothermophilus, Rhodobacter sphaeroides, Rhodomonas palustri,Streptococcus lactis, Pseudomonas purrocinia (ATCC 15958) or Pseudomonasfluorescens (NRRL B-11); Myxococcus, e.g., M. virescens.

Recombinantly produced peroxidases derived from Coprinus sp., inparticular C. macrorhizus or C. cinereus are described in WO 92/16634.Variants thereof are described in WO 94/12621.

Haloperoxidase

The enzyme exhibiting peroxidase activity may be any haloperoxidase.Haloperoxidases are widespread in nature and are known to be produced bymammals, plants, algae, lichen, bacteria, and fungi. There are threetypes of haloperoxidases, classified according to their specificity forhalide ions: Chloroperoxidases (E.C. 1.11.1.10) which catalyze thechlorination, bromination and iodination of compounds; bromoperoxidaseswhich show specificity for bromide and iodide ions; and iodoperoxidases(E.C. 1.11.1.8) which solely catalyze the oxidation of iodide ions.

Haloperoxidases include the haloperoxidase from Curvularia, inparticular, C. verruculosa, such as, C. verruculosa CBS 147.63 or C.verruculosa CBS 444.70. Curvularia haloperoxidase and recombinantproduction hereof is described in WO97/04102.

Bromide peroxidase has been isolated from algae (see U.S. Pat. No.4,937,192). Haloperoxidases are also described in U.S. Pat. No.6,372,465 (Novozymes A/S).

In a preferred embodiment, the haloperoxidase is a chloroperoxidase(E.C.1.11.1.10). Chloroperoxidases are known in the art and may beobtained from Streptomyces aureofaciens, Streptomyces lividans,Pseudomonas fluorescens, Caldariomyces fumago, Curvularia inaequalis,and Corallina officinalis. A preferred chloroperoxidase is thechloroperoxidase from Caldariomyces fumago (available from SIGMA,C-0278).

Haloperoxidases containing a vanadium prosthetic group are known toinclude at least two types of fungal chloroperoxidases from Curvulariainaequalis (van Schijndel et al., 1993, Biochimica Biophysica Acta1161:249-256; Simons et al., 1995, European Journal of Biochemistry 229:566-574; WO 95/27046) and Curvularia verruculosa (WO 97/04102) orPhaeotrichoconis crotalariae haloperoxidase (WO 2001/079461).

Lipoxygenase (LOX)

The enzyme exhibiting peroxidase activity may be any lipoxygenase (LOX).Lipoxygenases are classified as EC 1.13.11.12, which is an enzyme thatcatalyzes the oxygenation of polyunsaturated fatty acids, especiallycis,cis-1,4-dienes, e.g., linoleic acid and produces a hydroperoxide.But also other substrates may be oxidized, e.g., monounsaturated fattyacids. Microbial lipoxygenases may be derived from, e.g., Saccharomycescerevisiae, Thermoactinomyces vulgaris, Fusarium oxysporum, Fusariumproliferatum, Thermomyces lanuginosus, Pyricularia oryzae, and strainsof Geotrichum. The preparation of a lipoxygenase derived fromGaeumannomyces graminis is described in Examples 3-4 of WO 02/20730. Theexpression in Aspergillus oryzae of a lipoxygenase derived fromMagnaporthe salvinii is described in Example 2 of WO 02/086114, and thisenzyme can be purified using standard methods, e.g., as described inExample 4 of WO 02/20730.

Lipoxygenase (LOX) may also be extracted from plant seeds, such assoybean, pea, chickpea, and kidney bean. Alternatively, lipoxygenase maybe obtained from mammalian cells, e.g., rabbit reticulocytes.

Cellulases or Cellulolytic Enzymes

The term “cellulases” or “cellulolytic enzymes” as used herein areunderstood as comprising the cellobiohydrolases (EC 3.2.1.91), e.g.,cellobiohydrolase I and cellobiohydrolase II, as well as theendo-glucanases (EC 3.2.1.4), and beta-glucosidases (EC 3.2.1.21).

In order to be efficient, the digestion of cellulose and hemicelluloserequires several types of enzymes acting cooperatively. At least threecategories of enzymes are important to convert cellulose intofermentable sugars: endo-glucanases (EC 3.2.1.4) cut cellulose chains atrandom; cellobiohydrolases (EC 3.2.1.91) cleave cellobiosyl units fromthe cellulose chain ends and beta-glucosidases (EC 3.2.1.21) convertcellobiose and soluble cellodextrins into glucose. Among these threecategories of enzymes involved in the biodegradation of cellulose,cellobiohydrolases are the key enzymes for the degradation of nativecrystalline cellulose. The term “cellobiohydrolase I” is defined hereinas a cellulose 1,4-beta-cellobiosidase (also referred to asexo-glucanase, exo-cellobiohydrolase or 1,4-beta-cellobiohydrolase)activity, as defined in the enzyme class EC 3.2.1.91, which catalyzesthe hydrolysis of 1,4-beta-D-glucosidic linkages in cellulose andcellotetraose, by the release of cellobiose from the non-reducing endsof the chains. The definition of the term “cellobiohydrolase IIactivity” is identical, except that cellobiohydrolase II attacks fromthe reducing ends of the chains.

Endoglucanases (EC No. 3.2.1.4) catalyze endo hydrolysis of1,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (suchas carboxy methyl cellulose and hydroxy ethyl cellulose), lichenin,beta-1,4 bonds in mixed beta-1,3 glucans such as cereal beta-D-glucansor xyloglucans and other plant material containing cellulosic parts. Theauthorized name is endo-1,4-beta-D-glucan 4-glucano hydrolase, but theabbreviated term endoglucanase is used in the present specification.

The cellulases or cellulolytic enzymes may comprise acarbohydrate-binding module (CBM) which enhances the binding of theenzyme to a cellulose-containing fiber and increases the efficacy of thecatalytic active part of the enzyme. A CBM is defined as contiguousamino acid sequence within a carbohydrate-active enzyme with a discreetfold having carbohydrate-binding activity. For further information onCBMs see, e.g., the CAZy internet server (Supra) or Tomme et al., 1995,in Enzymatic Degradation of Insoluble Polysaccharides (Saddler & Penner,eds.), Cellulose-binding domains: classification and properties. pp.142-163, American Chemical Society, Washington.

The cellulase activity may, in a preferred embodiment, be derived from afungal source, such as a strain of the genus Trichoderma, preferably astrain of Trichoderma reesei; a strain of the genus Humicola, such as astrain of Humicola insolens; or a strain of Chrysosporium, preferably astrain of Chrysosporium lucknowense.

In a preferred embodiment cellulase or cellulolytic enzyme preparationis a composition concerned in co-pending application U.S. provisionalapplication No. 60/941,251, which is hereby incorporated by reference.In a preferred embodiment the cellulase or cellulolytic enzymepreparation comprising a polypeptide having cellulolytic enhancingactivity, preferably a family GH61A polypeptide, preferably onedisclosed in WO 2005/074656 (Novozymes). The cellulolytic enzymepreparation may further comprise a beta-glucosidase, such as abeta-glucosidase derived from a strain of the genus Trichoderma,Aspergillus, or Penicillium, including the fusion protein havingbeta-glucosidase activity disclosed in U.S. provisional application No.60/832,511 (PCT/US2007/074038) (Novozymes). In a preferred embodimentthe cellulolytic enzyme preparation may also comprises a CBH II enzyme,preferably Thielavia terrestris cellobiohydrolase II (CEL6A). In anotherpreferred embodiment the cellulolytic enzyme preparation may alsocomprise cellulolytic enzymes, preferably one derived from Trichodermareesei or Humicola insolens.

In a specific embodiment the cellulolytic enzyme preparation may alsocomprise a polypeptide having cellulolytic enhancing activity (GH61A)disclosed in WO 2005/074656; a CBH II from Thielavia terrestriscellobiohydrolase II (CEL6A); and a beta-glucosidase (fusion proteindisclosed in U.S. provisional application No. 60/832,511 (orPCT/US2007/074038)), and cellulolytic enzymes derived from Trichodermareesei.

In another specific embodiment the cellulolytic enzyme preparation mayalso comprise a polypeptide having cellulolytic enhancing activity(GH61A) disclosed in WO 2005/074656; a beta-glucosidase (fusion proteindisclosed in U.S. provisional application No. 60/832,511 (orPCT/US2007/074038)), and cellulolytic enzymes derived from Trichodermareesei.

In preferred embodiments the cellulase or cellulolytic preparations areCellulolytic preparations A and B used in Examples 1 and 4,respectively, disclosed in U.S. provisional application No. 60/941,251.

In an embodiment the cellulase is the commercially available productCELLUCLAST® 1.5 L or CELLUZYME™ (Novozymes A/S, Denmark) or ACCELERASE™1000 (from Genencor Inc., USA).

A cellulase or cellulolytic enzyme may be added for hydrolyzing thepre-treated lignocellulose-containing material. The cellulase may bedosed in the range from 0.1-100 FPU per gram total solids (TS),preferably 0.5-50 FPU per gram TS, especially 1-20 FPU per gram TS.

Hemicellulases

Hemicellulose can be broken down by hemicellulases and/or acidhydrolysis to release its five and six carbon sugar components. In anembodiment of the invention the lignocellulose derived material may betreated with one or more hemicellulase.

Any hemicellulase suitable for use in hydrolyzing hemicellulose may beused. Preferred hemicellulases include xylanases, arabinofuranosidases,acetyl xylan esterase, feruloyl esterase, glucuronidases,endo-galactanase, mannases, endo or exo arabinases, exo-galactanases,and mixtures of two or more thereof. Preferably, the hemicellulase foruse in the present invention is an exo-acting hemicellulase, and morepreferably, the hemicellulase is an exo-acting hemicellulase which hasthe ability to hydrolyze hemicellulose under acidic conditions of belowpH 7, preferably pH 3-7. An example of hemicellulase suitable for use inthe present invention includes VISCOZYME™ (available from Novozymes A/S,Denmark).

Arabinofuranosidase (EC 3.2.1.55) catalyzes the hydrolysis of terminalnon-reducing alpha-L-arabinofuranoside residues in alpha-L-arabinosides.

Galactanase (EC 3.2.1.89), arabinogalactan endo-1,4-beta-galactosidase,catalyzes the endohydrolysis of 1,4-D-galactosidic linkages inarabinogalactans.

Pectinase (EC 3.2.1.15) catalyzes the hydrolysis of1,4-alpha-D-galactosiduronic linkages in pectate and othergalacturonans.

Xyloglucanase catalyzes the hydrolysis of xyloglucan.

The hemicellulase may be added in an amount effective to hydrolyzehemicellulose, such as, in amounts from about 0.001 to 0.5 wt. % oftotal solids (TS), more preferably from about 0.05 to 0.5 wt. % of TS.

Alpha-Amylase

According to the invention an alpha-amylase may be used. In a preferredembodiment the alpha-amylase is an acid alpha-amylase, e.g., fungal acidalpha-amylase or bacterial acid alpha-amylase. The term “acidalpha-amylase” means an alpha-amylase (E.C. 3.2.1.1) which added in aneffective amount has activity optimum at a pH in the range of 3 to 7,preferably from 3.5 to 6, or more preferably from 4-5.

Bacterial Alpha-Amylase

According to the invention the bacterial alpha-amylase is preferablyderived from the genus Bacillus.

In a preferred embodiment the Bacillus alpha-amylase is derived from astrain of B. licheniformis, B. amyloliquefaciens, B. subtilis or B.stearothermophilus, but may also be derived from other Bacillus sp.Specific examples of contemplated alpha-amylases include the Bacilluslicheniformis alpha-amylase shown in SEQ ID NO: 4 in WO 99/19467, theBacillus amyloliquefaciens alpha-amylase SEQ ID NO: 5 in WO 99/19467 andthe Bacillus stearothermophilus alpha-amylase shown in SEQ ID NO: 3 inWO 99/19467 (all sequences hereby incorporated by reference). In anembodiment of the invention the alpha-amylase may be an enzyme having adegree of identity of at least 60%, preferably at least 70%, morepreferred at least 80%, even more preferred at least 90%, such as atleast 95%, at least 96%, at least 97%, at least 98% or at least 99% toany of the sequences shown in SEQ ID NOS: 1, 2 or 3, respectively, in WO99/19467.

The Bacillus alpha-amylase may also be a variant and/or hybrid,especially one described in any of WO 96/23873, WO 96/23874, WO97/41213, WO 99/19467, WO 00/60059, and WO 02/10355 (all documentshereby incorporated by reference). Specifically contemplatedalpha-amylase variants are disclosed in U.S. Pat. No. 6,093,562,6,297,038 or 6,187,576 (hereby incorporated by reference) and includeBacillus stearothermophilus alpha-amylase (BSG alpha-amylase) variantshaving a deletion of one or two amino acid in positions R179 to G182,preferably a double deletion disclosed in WO 1996/023873—see e.g., page20, lines 1-10 (hereby incorporated by reference), preferablycorresponding to delta(181-182) compared to the wild-type BSGalpha-amylase amino acid sequence set forth in SEQ ID NO:3 disclosed inWO 99/19467 or deletion of amino acids R179 and G180 using SEQ ID NO:3in WO 99/19467 for numbering (which reference is hereby incorporated byreference). Even more preferred are Bacillus alpha-amylases, especiallyBacillus stearothermophilus alpha-amylase, which have a double deletioncorresponding to delta(181-182) and further comprise a N193Fsubstitution (also denoted 1181*+G182*+N193F) compared to the wild-typeBSG alpha-amylase amino acid sequence set forth in SEQ ID NO:3 disclosedin WO 99/19467.

Bacterial Hybrid Alpha-Amylase

A hybrid alpha-amylase specifically contemplated comprises 445C-terminal amino acid residues of the Bacillus licheniformisalpha-amylase (shown in SEQ ID NO: 4 of WO 99/19467) and the 37N-terminal amino acid residues of the alpha-amylase derived fromBacillus amyloliquefaciens (shown in SEQ ID NO: 5 of WO 99/19467), withone or more, especially all, of the following substitution:G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S (using the Bacilluslicheniformis numbering in SEQ ID NO: 4 of WO 99/19467). Also preferredare variants having one or more of the following mutations (orcorresponding mutations in other Bacillus alpha-amylase backbones):H154Y, A181T, N190F, A209V and Q264S and/or deletion of two residuesbetween positions 176 and 179, preferably deletion of E178 and G179(using the SEQ ID NO: 5 numbering of WO 99/19467).

Fungal Alpha-Amylase

Fungal alpha-amylases include alpha-amylases derived from a strain ofthe genus Aspergillus, such as, Aspergillus oryzae, Aspergillus nigerand Aspergillis kawachii alpha-amylases.

A preferred acidic fungal alpha-amylase is a Fungamyl-like alpha-amylasewhich is derived from a strain of Aspergillus oryzae. According to thepresent invention, the term “Fungamyl-like alpha-amylase” indicates analpha-amylase which exhibits a high identity, i.e., more than 70%, morethan 75%, more than 80%, more than 85% more than 90%, more than 95%,more than 96%, more than 97%, more than 98%, more than 99% or even 100%identity to the mature part of the amino acid sequence shown in SEQ IDNO: 10 in WO 96/23874.

Another preferred acidic alpha-amylase is derived from a strainAspergillus niger. In a preferred embodiment the acid fungalalpha-amylase is the one from A. niger disclosed as “AMYA_ASPNG” in theSwiss-prot/TeEMBL database under the primary accession no. P56271 anddescribed in WO 89/01969 (Example 3). A commercially available acidfungal alpha-amylase derived from Aspergillus niger is SP288 (availablefrom Novozymes A/S, Denmark).

Other contemplated wild-type alpha-amylases include those derived from astrain of the genera Rhizomucor and Meripilus, preferably a strain ofRhizomucor pusillus (WO 2004/055178 incorporated by reference) orMeripilus giganteus.

In a preferred embodiment the alpha-amylase is derived from Aspergilluskawachii and disclosed by Kaneko et al., 1996, J. Ferment. Bioeng. 81:292-298, “Molecular-cloning and determination of the nucleotide-sequenceof a gene encoding an acid-stable alpha-amylase from Aspergilluskawachii; and further as EMBL: #AB008370.

The fungal alpha-amylase may also be a wild-type enzyme comprising astarch-binding domain (SBD) and an alpha-amylase catalytic domain (i.e.,non-hybrid), or a variant thereof. In an embodiment the wild-typealpha-amylase is derived from a strain of Aspergillus kawachii.

Fungal Hybrid Alpha-Amylase

In a preferred embodiment the fungal acid alpha-amylase is a hybridalpha-amylase. Preferred examples of fungal hybrid alpha-amylasesinclude the ones disclosed in WO 2005/003311 or U.S. Publication No.2005/0054071 (Novozymes) or U.S. provisional application No. 60/638,614(Novozymes) which is hereby incorporated by reference. A hybridalpha-amylase may comprise an alpha-amylase catalytic domain (CD) and acarbohydrate-binding domain/module (CBM), such as a starch bindingdomain, and optional a linker.

Specific examples of contemplated hybrid alpha-amylases include thosedisclosed in Table 1 to 5 of the examples in U.S. provisionalapplication No. 60/638,614, including Fungamyl variant with catalyticdomain JA118 and Athelia rolfsii SBD (SEQ ID NO:100 in U.S. provisionalapplication No. 60/638,614), Rhizomucor pusillus alpha-amylase withAthelia rolfsii AMG linker and SBD (SEQ ID NO:101 in U.S. provisionalapplication No. 60/638,614), Rhizomucor pusillus alpha-amylase withAspergillus niger glucoamylase linker and SBD (which is disclosed inTable 5 as a combination of amino acid sequences SEQ ID NO:20, SEQ IDNO:72 and SEQ ID NO:96 in U.S. application Ser. No. 11/316,535 andfurther as SEQ ID NO: 13 herein) or as V039 in Table 5 in WO2006/069290, and Meripilus giganteus alpha-amylase with Athelia rolfsiiglucoamylase linker and SBD (SEQ ID NO: 102 in U.S. provisionalapplication No. 60/638,614). Other specifically contemplated hybridalpha-amylases are any of the ones listed in Tables 3, 4, 5, and 6 inExample 4 in U.S. application Ser. No. 11/316,535 and WO 2006/069290(hereby incorporated by reference).

Other specific examples of contemplated hybrid alpha-amylases includethose disclosed in U.S. Publication no. 2005/0054071, including thosedisclosed in Table 3 on page 15, such as Aspergillus niger alpha-amylasewith Aspergillus kawachii linker and starch binding domain.

Contemplated are also alpha-amylases which exhibit a high identity toany of above mention alpha-amylases, i.e., more than 70%, more than 75%,more than 80%, more than 85% more than 90%, more than 95%, more than96%, more than 97%, more than 98%, more than 99% or even 100% identityto the mature enzyme sequences.

An acid alpha-amylases may according to the invention be added in anamount of 0.1 to 10 AFAU/g DS, preferably 0.10 to 5 AFAU/g DS,especially 0.3 to 2 AFAU/g DS.

Commercial Alpha-Amylase Products

Preferred commercial compositions comprising alpha-amylase includeMYCOLASE from DSM, BAN™, TERMAMYL™ SC, FUNGAMYL™, LIQUOZYME™ X and SAN™SUPER, SAN™ EXTRA L (Novozymes A/S) and CLARASE™ L-40,000, DEX-LO™,SPEZYME™ FRED, SPEZYME™ AA, and SPEZYME™ DELTA AA (Genencor Int.), andthe acid fungal alpha-amylase sold under the trade name SP288 (availablefrom Novozymes A/S, Denmark).

Carbohydrate-Source Generating Enzyme

The term “carbohydrate-source generating enzyme” includes glucoamylase(being glucose generators), beta-amylase and maltogenic amylase (beingmaltose generators). A carbohydrate-source generating enzyme is capableof producing a carbohydrate that can be used as an energy-source by thefermenting organism(s) in question, for instance, when used in a processof the invention for producing a fermentation product, such as ethanol.The generated carbohydrate may be converted directly or indirectly tothe desired fermentation product, preferably ethanol. According to theinvention a mixture of carbohydrate-source generating enzymes may beused. Especially contemplated mixtures are mixtures of at least aglucoamylase and an alpha-amylase, especially an acid amylase, even morepreferred an acid fungal alpha-amylase. The ratio between acidic fungalalpha-amylase activity (AFAU) per glucoamylase activity (AGU) (AFAU perAGU) may in an embodiment of the invention be at least 0.1, inparticular at least 0.16, such as in the range from 0.12 to 0.50 ormore. Alternatively the ratio between acid fungal alpha-amylase activity(FAU-F) and glucoamylase activity (AGU) (i.e., FAU-F per AGU) may in anembodiment of the invention be between 0.1 and 100 AGU/FAU-F, inparticular between 2 and 50 AGU/FAU-F, such as in the range from 10-40AGU/FAU-F.

Glucoamylase

A glucoamylase used according to the invention may be derived from anysuitable source, e.g., derived from a microorganism or a plant.Preferred glucoamylases are of fungal or bacterial origin, selected fromthe group consisting of Aspergillus glucoamylases, in particular A.niger G1 or G2 glucoamylase (Boel et al., 1984, EMBO J. 3(5):1097-1102), or variants thereof, such as those disclosed in WO 92/00381,WO 00/04136 and WO 01/04273 (from Novozymes, Denmark); the A. awamoriglucoamylase disclosed in WO 84/02921, A. oryzae glucoamylase (Agric.Biol. Chem., 1991, 55(4): 941-949), or variants or fragments thereof.Other Aspergillus glucoamylase variants include variants with enhancedthermal stability: G137A and G139A (Chen et al., 1996, Prot. Eng. 9:499-505); D257E and D293E/Q (Chen et al., 1995, Prot. Eng. 8: 575-582);N182 (Chen et al., 1994, Biochem. J. 301: 275-281); disulphide bonds,A246C (Fierobe et al., 1996, Biochemistry 35: 8698-8704; andintroduction of Pro residues in position A435 and S436 (Li et al., 1997,Protein Eng. 10: 1199-1204.

Other glucoamylases include Athelia rolfsii (previously denotedCorticium rolfsi) glucoamylase (see U.S. Pat. No. 4,727,026 and Nagasakaet al., 1998, “Purification and properties of the raw-starch-degradingglucoamylases from Corticium rolfsii, Appl Microbiol Biotechnol 50:323-330), Talaromyces glucoamylases, in particular derived fromTalaromyces emersonii (WO 99/28448), Talaromyces leycettanus (U.S. Pat.No. Re. 32,153), Talaromyces duponti, Talaromyces thermophilus (U.S.Pat. No. 4,587,215).

Bacterial glucoamylases contemplated include glucoamylases from thegenus Clostridium, in particular C. thermoamylolyticum (EP 135,138), andC. thermohydrosulfuricum (WO 86/01831) and Trametes cingulata disclosedin WO 2006/069289 (which is hereby incorporated by reference).

Also hybrid glucoamylase are contemplated according to the invention.Examples the hybrid glucoamylases disclosed in WO 2005/045018. Specificexamples include the hybrid glucoamylase disclosed in Table 1 and 4 ofExample 1 (which hybrids are hereby incorporated by reference.).

Contemplated are also glucoamylases which exhibit a high identity to anyof above mention glucoamylases, i.e., more than 70%, more than 75%, morethan 80%, more than 85% more than 90%, more than 95%, more than 96%,more than 97%, more than 98%, more than 99% or even 100% identity to themature enzymes sequences.

Commercially available compositions comprising glucoamylase include AMG200L; AMG 300 L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™FUEL, SPIRIZYME™ B4U and AMG™ E (from Novozymes A/S); OPTIDEX™ 300 (fromGenencor Int.); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900,G-ZYME™ and G990 ZR (from Genencor Int.).

Glucoamylases may in an embodiment be added in an amount of 0.02-20AGU/g DS, preferably 0.1-10 AGU/g DS, especially between 1-5 AGU/g DS,such as 0.5 AGU/g DS.

Beta-Amylase

At least according to the invention beta-amylase (E.C 3.2.1.2) is thename traditionally given to exo-acting maltogenic amylases, whichcatalyze the hydrolysis of 1,4-alpha-glucosidic linkages in amylose,amylopectin and related glucose polymers. Maltose units are successivelyremoved from the non-reducing chain ends in a step-wise manner until themolecule is degraded or, in the case of amylopectin, until a branchpoint is reached. The maltose released has the beta anomericconfiguration, hence the name beta-amylase.

Beta-amylases have been isolated from various plants and microorganisms(Fogarty and Kelly, 1979, Progress in Industrial Microbiology 15:112-115). These beta-amylases are characterized by having optimumtemperatures in the range from 40° C. to 65° C. and optimum pH in therange from 4.5 to 7. A commercially available beta-amylase from barleyis NOVOZYM™ WBA from Novozymes A/S, Denmark and SPEZYME™ BBA 1500 fromGenencor Int., USA.

Maltogenic Amylase

The amylase may also be a maltogenic alpha-amylase. A “maltogenicalpha-amylase” (glucan 1,4-alpha-maltohydrolase, E.C. 3.2.1.133) is ableto hydrolyze amylose and amylopectin to maltose in thealpha-configuration. A maltogenic amylase from Bacillusstearothermophilus strain NCIB 11837 is commercially available fromNovozymes A/S. Maltogenic alpha-amylases are described in U.S. Pat. Nos.4,598,048, 4,604,355 and 6,162,628, which are hereby incorporated byreference.

The maltogenic amylase may in a preferred embodiment be added in anamount of 0.05-5 mg total protein/gram DS or 0.05-5 MANU/g DS.

Proteases

The protease may according to the invention be any protease. In apreferred embodiment the protease is an acid protease of microbialorigin, preferably of fungal or bacterial origin.

Suitable proteases include microbial proteases, such as fungal andbacterial proteases. Preferred proteases are acidic proteases, i.e.,proteases characterized by the ability to hydrolyze proteins underacidic conditions below pH 7.

Contemplated acid fungal proteases include fungal proteases derived fromAspergillus, Mucor, Rhizopus, Candida, Coriolus, Endothia, Enthomophtra,Irpex, Penicillium, Scierotiumand, and Torulopsis. Especiallycontemplated are proteases derived from Aspergillus niger (see, e.g.,Koaze et al., 1964, Agr. Biol. Chem. Japan 28: 216), Aspergillus saitoi(see, e.g., Yoshida, 1954, J. Agr. Chem. Soc. Japan 28: 66), Aspergillusawamori (Hayashida et al., 1977, Agric. Biol. Chem. 42(5): 927-933,Aspergillus aculeatus (WO 95/02044), or Aspergillus oryzae, such as thepepA protease; and acidic proteases from Mucor pusillus or Mucor miehei.

Contemplated are also neutral or alkaline proteases, such as a proteasederived from a strain of Bacillus. A particular protease contemplatedfor the invention is derived from Bacillus amyloliquefaciens and has thesequence obtainable at Swissprot as Accession No. P06832. Alsocontemplated are the proteases having at least 90% identity to aminoacid sequence obtainable at Swissprot as Accession No. P06832 such as atleast 92%, at least 95%, at least 96%, at least 97%, at least 98%, orparticularly at least 99% identity.

Further contemplated are the proteases having at least 90% identity toamino acid sequence disclosed as SEQ.ID.NO:1 in the WO 2003/048353 suchas at 92%, at least 95%, at least 96%, at least 97%, at least 98%, orparticularly at least 99% identity.

Also contemplated are papain-like proteases such as proteases withinE.C. 3.4.22.* (cysteine protease), such as EC 3.4.22.2 (papain), EC3.4.22.6 (chymopapain), EC 3.4.22.7 (asclepain), EC 3.4.22.14(actinidain), EC 3.4.22.15 (cathepsin L), EC 3.4.22.25 (glycylendopeptidase) and EC 3.4.22.30 (caricain).

Proteases may be added in the amounts of 0.1-1000 AU/kg dm, preferably1-100 AU/kg DS and most preferably 5-25 AU/kg DS.

Use

In the third aspect the invention relates to the use of one or morephenolic compound oxidizing enzymes and/or enzymes exhibiting peroxidaseactivity for detoxifying pre-treated lignocellulose-containing material.

In a preferably embodiment the phenolic compound oxidizing enzyme may beselected from the group comprising catechol oxidase (EC 1.10.3.1),laccase (EC 1.10.3.2), o-aminophenol oxidase (EC 1.10.3.4); andmonophenol monooxygenase (EC 1.14.18.1) for detoxifying pre-treatedlignocellulose-containing material.

In another preferred embodiment the enzyme exhibiting peroxidaseactivity may be selected from the group comprising peroxidase (EC1.11.1.7), haloperoxidase (EC1.11.1.8 and EC 1.11.1.10); ligninperoxidase (EC 1.11.1.14); manganese peroxidase (EC 1.11.1.13); andlipoxygenase (EC.1.13.11.12) for detoxifying pre-treatedlignocellulose-containing material.

The detoxification may be part of a fermentation product productionprocess of the invention.

Materials & Methods Enzymes: Laccase PpL:

Laccase derived from Polyporus pinsitus disclosed in WO 1996/000290(Novozymes).

Laccase MtL:

Laccase derived from Myceliopthora thermophila disclosed in WO1995/033836 (Novozymes).

Laccase CcL:

Laccase derived from Coprinus cinereus disclosed in WO 97/08325(Novozymes)

Peroxidase CcP:

Peroxidase is derived from Coprinus cinereus disclosed in Petersen etal., 1994, FEBS Letters 339: 291-296.

Cellulolutic Preparation A:

Cellulolytic composition comprising a polypeptide having cellulolyticenhancing activity (GH61A) disclosed in WO 2005/074656; abeta-glucosidase (fusion protein disclosed in U.S. provisionalapplication No. 60/832,511), Thielavia terrestris cellobiohydrolase II(CEL6A), and cellulolytic enzymes preparation derived from Trichodermareesei. Cellulase preparation A is disclosed in U.S. provisionalapplication No. 60/941,251.

Cellulolytic Preparation B:

Cellulolytic composition comprising a polypeptide having cellulolyticenhancing activity (GH61A) disclosed in WO 2005/074656; abeta-glucosidase (fusion protein disclosed in U.S. provisionalapplication No. 60/832,511); and cellulolytic enzymes preparationderived from Trichoderma reesei. Cellulase preparation A is disclosed inU.S. provisional application No. 60/941,251.

Yeast:

RED STAR™ available from Red Star/Lesaffre, USA

Pre-Treated Corn Stover:

dilute acid-catalyzed steam explosion corn stover (28.6% DS) wasobtained from NREL (National Renewable Research Laboratory, USA).

Determination of Identity

The relatedness between two amino acid sequences or between twonucleotide sequences is described by the parameter “identity”.

The degree of identity between two amino acid sequences may bedetermined by the Clustal method (Higgins, 1989, CABIOS 5: 151-153)using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.)with an identity table and the following multiple alignment parameters:Gap penalty of 10 and gap length penalty of 10. Pairwise alignmentparameters are Ktuple=1, gap penalty=3, windows=5, and diagonals=5.

The degree of identity between two nucleotide sequences may bedetermined by the Wilbur-Lipman method (Wilbur and Lipman, 1983,Proceedings of the National Academy of Science USA 80: 726-730) usingthe LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.) with anidentity table and the following multiple alignment parameters: Gappenalty of 10 and gap length penalty of 10. Pairwise alignmentparameters are Ktuple=3, gap penalty=3, and windows=20.

Determination of Laccase Activity (LACU)

Laccase activity is determined from the oxidation of syringaldazin underaerobic conditions. The violet color produced is photometered at 530 nm.The analytical conditions are 19 mM syringaldazin, 23.2 mM acetatebuffer, pH 5.5, 30° C., 1 min. reaction time.

1 laccase unit (LACU) is the amount of enzyme that catalyzes theconversion of 1.0 micromole syringaldazin per minute at theseconditions.

Determination of Peroxidase Activity (PODU)

One peroxidase unit (PODU) is defined as the amount of enzyme which,under standard conditions (i.e., pH 7.0; temperature 30° C.; reactiontime 3 minutes) catalyzes the conversion of 1 micromole hydrogenperoxide per minute. The activity is determined using an assay based onABTS® (2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate)) as thechromophore, the greenish-blue colour produced being photometered at 418nm. A folder AF 279/2 describing this analytical method in more detailis available upon request to Novozymes A/S, Denmark, which folder ishereby included by reference.

Glucoamylase Activity

Glucoamylase activity may be measured in Glucoamylase Units (AGU).

Glucoamylase Activity (AGU)

The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme,which hydrolyzes 1 micromole maltose per minute under the standardconditions 37° C., pH 4.3, substrate: maltose 23.2 mM, buffer: acetate0.1 M, reaction time 5 minutes.

An autoanalyzer system may be used. Mutarotase is added to the glucosedehydrogenase reagent so that any alpha-D-glucose present is turned intobeta-D-glucose. Glucose dehydrogenase reacts specifically withbeta-D-glucose in the reaction mentioned above, forming NADH which isdetermined using a photometer at 340 nm as a measure of the originalglucose concentration.

AMG incubation: Substrate: maltose 23.2 mM Buffer: acetate 0.1M pH: 4.30± 0.05 Incubation temperature: 37° C. ± 1 Reaction time: 5 minutesEnzyme working range: 0.5-4.0 AGU/mL

Color reaction: GlucDH: 430 U/L Mutarotase: 9 U/L NAD: 0.21 mM Buffer:phosphate 0.12M; 0.15M NaCl pH: 7.60 ± 0.05 Incubation temperature: 37°C. ± 1 Reaction time: 5 minutes Wavelength: 340 nm

A folder (EB-SM-0131.02/01) describing this analytical method in moredetail is available on request from Novozymes A/S, Denmark, which folderis hereby included by reference.

Alpha-Amylase Activity (KNU)

The alpha-amylase activity may be determined using potato starch assubstrate. This method is based on the break-down of modified potatostarch by the enzyme, and the reaction is followed by mixing samples ofthe starch/enzyme solution with an iodine solution. Initially, ablackish-blue color is formed, but during the break-down of the starchthe blue color gets weaker and gradually turns into a reddish-brown,which is compared to a colored glass standard.

One Kilo Novo alpha amylase Unit (KNU) is defined as the amount ofenzyme which, under standard conditions (i.e., at 37° C.+/−0.05; 0.0003M Ca²⁺; and pH 5.6) dextrinizes 5260 mg starch dry substance MerckAmylum solubile.

A folder EB-SM-0009.02/01 describing this analytical method in moredetail is available upon request to Novozymes A/S, Denmark, which folderis hereby included by reference.

Acid Alpha-Amylase Activity

When used according to the present invention the activity of an acidalpha-amylase may be measured in AFAU (Acid Fungal Alpha-amylase Units)or FAU-F (Fungal Alpha-Amylase Units).

Acid Alpha-Amylase Activity (AFAU)

Acid alpha-amylase activity may be measured in AFAU (Acid FungalAlpha-amylase Units), which are determined relative to an enzymestandard. 1 AFAU is defined as the amount of enzyme which degrades 5.260mg starch dry matter per hour under the below mentioned standardconditions.

Acid alpha-amylase, an endo-alpha-amylase(1,4-alpha-D-glucan-glucanohydrolase, E.C. 3.2.1.1) hydrolyzesalpha-1,4-glucosidic bonds in the inner regions of the starch moleculeto form dextrins and oligosaccharides with different chain lengths. Theintensity of color formed with iodine is directly proportional to theconcentration of starch. Amylase activity is determined using reversecolorimetry as a reduction in the concentration of starch under thespecified analytical conditions.

Standard Conditions/Reaction Conditions:

-   -   Substrate: Soluble starch, approx. 0.17 g/L    -   Buffer: Citrate, approx. 0.03 M    -   Iodine (12): 0.03 g/L    -   CaC₂: 1.85 mM    -   pH: 2.50±0.05    -   Incubation temperature: 40° C.    -   Reaction time: 23 seconds    -   Wavelength: 590 nm    -   Enzyme concentration: 0.025 AFAU/mL    -   Enzyme working range: 0.01-0.04 AFAU/mL

A folder EB-SM-0259.02/01 describing this analytical method in moredetail is available upon request to Novozymes A/S, Denmark, which folderis hereby included by reference.

Determination of FAU-F

FAU-F Fungal Alpha-Amylase Units (Fungamyl) is measured relative to anenzyme standard of a declared strength.

Reaction conditions Temperature 37° C. pH 7.15 Wavelength 405 nmReaction time 5 min Measuring time 2 min

A folder (EB-SM-0216.02) describing this standard method in more detailis available on request from Novozymes A/S, Denmark, which folder ishereby included by reference.

Measurement of Cellulase Activity Using Filter Paper Assay (FPUAssay) 1. Source of Method

1.1 The method is disclosed in a document entitled “Measurement ofCellulase Activities” by Adney and Baker, 1996, Laboratory AnalyticalProcedure, LAP-006, National Renewable Energy Laboratory (NREL). It isbased on the IUPAC method for measuring cellulase activity (Ghose, 1987,Measurement of Cellulase Activities, Pure & Appl. Chem. 59: 257-268.

2. Procedure

2.1 The method is carried out as described by Adney and Baker, 1996,supra, except for the use of a 96 well plates to read the absorbancevalues after color development, as described below.

2.2 Enzyme Assay Tubes:

-   2.2.1 A rolled filter paper strip (#1 Whatman; 1×6 cm; 50 mg) is    added to the bottom of a test tube (13×100 mm).-   2.2.2 To the tube is added 1.0 mL of 0.05 M Na-citrate buffer (pH    4.80).-   2.2.3 The tubes containing filter paper and buffer are incubated 5    min. at 50° C. (±0.1° C.) in a circulating water bath.-   2.2.4 Following incubation, 0.5 mL of enzyme dilution in citrate    buffer is added to the tube.    -   Enzyme dilutions are designed to produce values slightly above        and below the target value of 2.0 mg glucose.-   2.2.5 The tube contents are mixed by gently vortexing for 3 seconds.-   2.2.6 After vortexing, the tubes are incubated for 60 mins. at    50° C. (±0.1° C.) in a circulating water bath.-   2.2.7 Immediately following the 60 min. incubation, the tubes are    removed from the water bath, and 3.0 mL of DNS reagent is added to    each tube to stop the reaction. The tubes are vortexed 3 seconds to    mix.

2.3 Blank and Controls

-   2.3.1 A reagent blank is prepared by adding 1.5 mL of citrate buffer    to a test tube.-   2.3.2 A substrate control is prepared by placing a rolled filter    paper strip into the bottom of a test tube, and adding 1.5 mL of    citrate buffer.-   2.3.3 Enzyme controls are prepared for each enzyme dilution by    mixing 1.0 mL of citrate buffer with 0.5 mL of the appropriate    enzyme dilution.-   2.3.4 The reagent blank, substrate control, and enzyme controls are    assayed in the same manner as the enzyme assay tubes, and done along    with them.

2.4 Glucose Standards

-   2.4.1 A 100 mL stock solution of glucose (10.0 mg/mL) is prepared,    and 5 mL aliquots are frozen. Prior to use, aliquots are thawed and    vortexed to mix.-   2.4.2 Dilutions of the stock solution are made in citrate buffer as    follows:    G1=1.0 mL stock+0.5 mL buffer=6.7 mg/mL=3.3 mg/0.5 mL    G2=0.75 mL stock+0.75 mL buffer=5.0 mg/mL=2.5 mg/0.5 mL    G3=0.5 mL stock+1.0 mL buffer=3.3 mg/mL=1.7 mg/0.5 mL    G4=0.2 mL stock+0.8 mL buffer=2.0 mg/mL=1.0 mg/0.5 mL-   2.4.3 Glucose standard tubes are prepared by adding 0.5 mL of each    dilution to 1.0 mL of citrate buffer.-   2.4.4 The glucose standard tubes are assayed in the same manner as    the enzyme assay tubes, and done along with them.

2.5 Color Development

-   2.5.1 Following the 60 min. incubation and addition of DNS, the    tubes are all boiled together for 5 mins. in a water bath.-   2.5.2 After boiling, they are immediately cooled in an ice/water    bath.-   2.5.3 When cool, the tubes are briefly vortexed, and the pulp is    allowed to settle. Then each tube is diluted by adding 50 microL    from the tube to 200 microL of ddH₂O in a 96-well plate. Each well    is mixed, and the absorbance is read at 540 nm.

2.6 Calculations (Examples are Given in the NREL Document)

-   2.6.1 A glucose standard curve is prepared by graphing glucose    concentration (mg/0.5 mL) for the four standards (G1-G4) vs. A₅₄₀.    This is fitted using a linear regression (Prism Software), and the    equation for the line is used to determine the glucose produced for    each of the enzyme assay tubes.-   2.6.2 A plot of glucose produced (mg/0.5 mL) vs. total enzyme    dilution is prepared, with the Y-axis (enzyme dilution) being on a    log scale.-   2.6.3 A line is drawn between the enzyme dilution that produced just    above 2.0 mg glucose and the dilution that produced just below that.    From this line, it is determined the enzyme dilution that would have    produced exactly 2.0 mg of glucose.-   2.6.4 The Filter Paper Units/mL (FPU/mL) are calculated as follows:    FPU/mL=0.37/enzyme dilution producing 2.0 mg glucose

Protease Assay Method—AU(RH)

The proteolytic activity may be determined with denatured hemoglobin assubstrate. In the Anson-Hemoglobin method for the determination ofproteolytic activity denatured hemoglobin is digested, and theundigested hemoglobin is precipitated with trichloroacetic acid (TCA).The amount of TCA soluble product is determined with phenol reagent,which gives a blue color with tyrosine and tryptophan.

One Anson Unit (AU-RH) is defined as the amount of enzyme which understandard conditions (i.e., 25° C., pH 5.5 and 10 min. reaction time)digests hemoglobin at an initial rate such that there is liberated perminute an amount of TCA soluble product which gives the same color withphenol reagent as one milliequivalent of tyrosine.

The AU(RH) method is described in EAL-SM-0350 and is available fromNovozymes A/S Denmark on request.

EXAMPLES Example 1 Effect of Laccase on Ethanol Yield During EnzymaticHydrolysis

Dilute acid-catalyzed steam exploded pre-treated corn stover wasobtained from NREL. The pre-treated corn stover (15% DS) was hydrolyzedat pH 4.5, 50° C. for 75 hours with Cellulolytic preparation A (5 FPU/gDS) with and without Laccase PpL (20-30 LACU/g DS). The enzymatictreatment was carried out with open lid so that consistent air flow wasprovided during the treatment. Evaporation was control by daily watersupplement based on weight loss. The enzyme treated samples were usedfor ethanol fermentation at 32° C. for up to 88 hours in a closed vesselwhere a needle was punched in the cap, with yeast (RED STAR™) at initialdosage of 0.2 g/L. The fermentation mixture also contains YPU (5 g/Lyeast extract, 5 g/L peptone and 10 g/L Urea). After 25 hoursfermentation, the laccase treated sample resulted in 10 g/L ethanolproduction, whereas the non-laccase treated sample resulted in noethanol yield. With initial yeast dosage of 1.6 g/L, about 10-foldhigher ethanol production was observed with laccase treated sample after25 hours fermentation (18.87 g/L for laccase treated samples vs 1.83 fornon-laccase treated sample).

Example 2 Effect of Laccase on Ethanol Yield During Fermentation

The pre-treated corn stover (28.6% DS) used was the same as inExample 1. The pre-treated corn stover (15% DS) was hydrolyzed withCellulolytic preparation A (5 FPU/g DS) at pH 4.5, 50° C. for 75 hoursin the absence of laccase with covered lid. After the enzymatichydrolysis, material was fermented at 32° C. for up to 88 hours in aclosed vessel where a needle was punched in the cap, with yeast (REDSTAR™) at dosage of 1.6 g/L with and without laccase PpL (20-30 LACU/gDS). The fermentation mixture also contains YPU (5 g/L yeast extract, 5g/L peptone and 10 g/L Urea). After 25 hours, the ethanol production wasdoubled in the sample where laccase was present during fermentation(4.59 g/L laccase treated vs 1.83 non-laccase treated).

Example 3 Effect of Oxidoreductases Treatment in Between Hydrolysis andYeast Fermentation

The pre-treated corn stover (28.6% DS) used was the same as inExample 1. Enzymatic hydrolysis was carried out with pre-treated cornstover (15% DS) at pH 4.5, 50° C. for 72 hours in the absence of laccasewith covered lid. Oxidoreductase treatment was conducted at 50° C. forone or two hours before fermentation at 32° C. (up to 48 hours) withyeast (RED STAR™) at dosage of 0.5 g/L in a closed vessel where a needlewas punched in the cap. During oxidoreductase treatment, lids wereopened every 20 minutes. The fermentation mixture also contains YPU (5g/L yeast extract, 5 g/l peptone and 10 g/L Urea). Up to 40% increase inethanol production was observed with Laccase PpL (4.5-30 LACU/g DS). Upto 27% increase in ethanol production was observed with Laccase MtL(0.5-30 LACU/g DS). About 27% increase in ethanol production wasobserved with Laccase CcL (36 LACU/g DS). About 40% increase in ethanolproduction was observed with peroxidase CcP in the presence of 5% H₂O₂.

Example 4 Laccase-Mediated Improvement of PCS Enzymatic HydrolysisCollection of Pretreatment Liquor:

Liquor was collected from both neutral, steam exploded corn stover andfrom acid, steam exploded corn stover. Each PCS was slurried indeionized water to a final total solids (TS) level of 15 wt. % withmixing at ambient temperature for 1 hour. Each slurry was stored at 4°C. for 16-20 hours. Slurries were then mixed at ambient temperature for1 hour, and liquor was collected by vacuum filtration through a glassfiber filter (Whatman GF/D). Sodium azide was added to a finalconcentration of 0.02% w/w. The pH of each was adjusted to 5.0. Aftermixing for 1 hour at ambient temperature, liquor was vacuum filteredthrough a 0.2 micro m membrane and stored at 4° C.

Laccase Treatment:

All PCS liquors were adjusted to pH 5.0. Laccase MtL was dosed into PCSliquor to a final concentration of 100 ppm. The liquor containinglaccase was incubated alongside a negative control liquor for 18 hoursat 50° C. with 150 rpm of agitation. Any precipitated material wasremoved by centrifugation at 3000 rpm for 10 minutes prior to furthercharacterization.

Folin-Ciocalteu (FC) Method for Phenolics:

The method was modified from a published procedure (Singleton, Orthofer,and Lamuela-Raventos, 1999, Methods Enzymol. 299: 152-178). Catechol(Sigma #135011) calibration standards and sample dilutions were preparedin deionized water. Fifty microL of diluted sample or catecholcalibration standard were transferred to the wells of a microtiterplate. Deionized water (50 microL) was added to each well followed by 50microL/well of FC reagent (Sigma #F9252). The plate was incubated for 5minutes at ambient temperature. Sodium carbonate (15% w/v, 100 microL)was then added to each well and the plate was incubated for 30 minutesat ambient temperature in the dark. The absorbance at 770 nm of eachwell was collected. Unknown total phenolic concentrations werecalculated from the catechol standard curve by linear regressionanalysis in Microsoft Excel.

PCS Hydrolysis and Glucose Monitoring.

Washed PCS solids were slurried in the appropriate PCS liquor at a finalconcentration of 4% total solids and dosed with Cellulolytic preparationB (3 mg protein/g cellulose). Hydrolysis reactions were incubated at 50°C. with shaking (150 rpm) for 48 hours. Glucose concentrations weremonitored over time using an enzyme-coupled glucose assay.

TABLE 1 Effect of laccase treatment on soluble phenolics as measured byFC method Phenolics, mg/mL Liquor −laccase +laccase % Decrease NeutralPCS 2.30 ± 0.05 1.63 ± 0.01 29.1 Acid PCS 1.96 ± 0.02 1.34 ± 0.02 31.8

CONCLUSIONS

Enzymatic hydrolysis of PCS was improved by treating PCS liquor with alaccase. Laccase treatment of either neutral or acid PCS liquorsresulted in an 8-10% improvement in cellulose conversion.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure, including definitions will becontrolling.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1-20. (canceled)
 21. A process for producing a fermentation product fromlignocellulose-containing material, comprising the steps of: (a)pre-treating lignocellulose-containing material; (b) detoxifyingcomprising subjecting the pre-treated lignocellulose-containing materialto one or more phenolic compound oxidizing enzymes and/or one or moreenzymes exhibiting peroxidase activity; (c) hydrolyzing; and (d)fermenting using a fermenting organism, wherein the detoxification iscarried out simultaneously with hydrolysis or fermentation.
 22. Theprocess of claim 21, wherein the one or more phenolic compound oxidizingenzymes are selected from the group consisting of a catechol oxidase (EC1.10.3.1), laccase (EC 1.10.3.2), o-aminophenol oxidase (EC 1.10.3.4);and monophenol monooxygenase (EC 1.14.18.1).
 23. The process of claim21, wherein the enzyme exhibiting peroxidase activity is selected fromthe group consisting of a peroxidase (EC 1.11.1.7), haloperoxidase(EC1.11.1.8 and EC 1.11.1.10); lignin peroxidase (EC 1.11.1.14);manganese peroxidase (EC 1.11.1.13); and lipoxygenase (EC.1.13.11.12).24. The process of claim 21, wherein the solids after pre-treating thelignocellulose-containing material in step (a) are removed/separatedfrom the liquor before detoxification.
 25. The process of claim 21,wherein the lignocellulose-containing material is chemically,mechanically and/or biologically pre-treated in step (a).
 26. Theprocess of claim 21, wherein hydrolysis and/or fermentation is carriedout using one or more carbohydrases selected from the group consistingof cellulase, hemicellulase, amylase, protease, esterase, endoglucanase,beta-glucosidase, cellobiohydrolase, xylanase, alpha-amylase,alpha-glucosidase, glucoamylase, protease and lipase.
 27. The process ofclaim 21, wherein the fermenting organism is a yeast strain of the genusSaccharomyces.
 28. The process of claim 1, wherein the fermentationproduct is an alcohol.